Spectrophotometry Dr. Shareef SHAIK ASST. PROFESSOR Pharmacology
Content Introduction Beer-Lambert law Instrument Applications
Introduction 3 Body fluids such as blood, csf and urine contain organic and inorganic substances. Organic substances glucose, urea, uric acid etc. Inorganic substances potassium, calcium, phosphorus, chloride, etc. These substances are in an equilibrium as` the rate of production and loss are equal.
Introduction Normal concentration of these substances vary within very narrow limits. Because of small concentration of these substances and difficulty in isolating them photometric methods are frequently used.
Introdution 5 compounds absorb light radiation of a specific wavelength. the amount of light radiation absorbed by a sample is measured. The light absorption is directly related to the concentration of the compound in the sample.
Optical techniques Based on estimation of light absorbing nature of the substances 1.Calorimetry - can analyze only coloured compounds 2.Spectrophotometry both coloured compounds and colourless as well.
Optical techniques Based on estimation of light emitting nature of the substances 1.Flame photometry 2.Fluorimetry
Spectrum It is not 2G spectrum scam by which parliament was adjourned unendingly. The word most heard till recently How does our spectrum differs from 2G?
Spectrum In Latin spectrum means "image" or "apparition In the 17th century the word spectrum was introduced into optics, referring to the range of colors observed when white light was dispersed through a prism It has since been applied by analogy to many fields other than optics. Thus, one might talk about the spectrum of political opinion, or the spectrum of activity of a drug
Distribution of radiant energy Wave Number (cycles/cm) -Ray X-Ray UV Visible IR Microwave 200nm 400nm 800nm Wavelength (nm)
Spectrophotometry 11 Photometry is defined as measurement of light Modern instruments isolate a narrow wavelength range of spectrum for measurements. Those that use filters Filterphotometer prism or gratings - Spectrophotometer
Spectrophotometer An instrument which can measure the absorbance of a sample at a particular wavelength.
General principles 13 Light I M I Glass cell filled with concentration of solution (C)
Beer s law Absorbance of a solution is directly proportional to the concentration of a solution { A α C} or Transmittance of a solution decreases exponentially with the increase in concentration of the solution T = e -kc A = absorbance T = transmittance C = conc. of solution
Lambert s law Absorbance of a solution is directly proportional to the thickness of the optical path { A α t} or Transmittance of a solution decreases exponentially with the increase in thickness of the optical path T = e -kt A = absorbance T = transmittance C = conc. of solution t = thickness of the optical path
Beer Lambert Law If both these are combined, A α Ct or T = e -kct A = absorbance T = transmittance C = conc. of solution t = thickness of the optical path K = constant for the substance
Spectrophotometric analysis 17 Widely used method of quantitative and qualitative analysis Accurate and sensitive
Spectrophotometers Two types Single beam Double beam
Instruments 21 Light source provide a sufficient of light which is suitable for making a measurement. The light source typically yields a high output of polychromatic light over a wide range of the spectrum.
Light sources 1. Incandescent lamps 2. Laser sources
Incandescent lamps 23 UV light 1.Deuterium lamp 2.Hydrogen Gas Lamp 3.Mercury Lamp Visible light 1. Tungsten Lamp (wavelength ranges from 360 700nm)
Light Sources 24 Laser sources Laser - Light amplification by stimulated emission of radiation They are used as they provide intense light of narrow wavelength Allow beam diameters in the range of several microns Produce monochromatic light Wavelength output ranges from 550 1810 nm
Monochromator 25 A system for isolating radiant energy of a desired wavelength and excluding that of other wavelengths is called monochromator. Modern instruments isolate a narrow wavelength of the spectrum for measurements - Isolation of spectrum
Monochromator Glass filters (coloured glass) calorimeter Prisms or gratings Spectrophotometers A glass filter is not true monochromatic light as it transmits light over a wide range of wavelengths.
Dispersion of Polychromatic Light with a Prism Prism - Spray out the spectrum and choose the certain wavelength ( that you want by slit. Infrared m onochrom atic R ay P olychrom atic R ay PR ISM R ed O range Y ellow G reen B lue SLIT V iolet U ltraviolet Polychrom atic R ay M onochrom atic R ay
Slit 28 Slit this is to allow a narrow beam of selected monochromatic light to pass through the sample solution Improve its chromatic purity
Cuvette A small vessel used to hold a liquid sample to be analyzed. Shape round,square,rectangular Constructed from glass,silica quartz Square or rectangular cuvettes have plane- paralled optical light surfaces and a constant light path Most have a 1cm light path
Cuvette It is to be filled ¾ th of its height Quartz cuvette is used in UV analysis
Cuvette - care Must be clean and optically clear,as etching or deposits on the surface affect absorbance values Glass cuvete cleaned by copious rinsing with tap water and distilled water Cuvets may be cleaned by detergent or soaked in a mixture of conc HCL: water : ethanol (1:3:4)
Photodetector Devices that convert light into electric signal that is proportional to the number of photons striking its photosensitive surface. Photomultiplier tubes (PMT) are commonly used as detectors Photodiodes are used in modern instruments
Readout device The electrical signal from the PMT is amplified and then recorded by the galvanometer. Usually the recorders are calibrated in such a way that they directly give the absorbance or converted values of concentration. Now we have digital readout devices These operate on the principle of selective illumination of a portions of a bank of light-emitting diodes (LEDs)
Spectrophotometer Reading
Applications 35 In clinical diagnostic and research laboratories Quantitative estimation of different compounds in various biological fluids. E.g. Blood glucose, urea, cholesterol, creatinine, bilirubin and CSF protein etc. The absorption spectrum of a compound is characteristic for it Useful in identification and determination of purity and structure of a compound
UV Spectrophotometer Application Protein Amino Acids (aromatic) Pantothenic Acid Glucose Determination Enzyme Activity (Hexokinase)
Visible Spectrophotometer Application 37 Niacin Pyridoxine Vitamin B12 Metal Determination (Fe) Enzyme Activity (glucose oxidase)
Applications 38 38 1. Concentration measurement Prepare samples Make series of standard solutions of known concentrations
Applications 39 Measure the absorption of the unknown from the standard plot, read the related concentration
Applications 40 5. Detection of Functional Groups Absence of a band at particular wavelength regarded as an evidence for absence of particular group
Applications 41 2. Detection of Impurities UV absorption spectrophotometry is one of the best methods for determination of impurities in organic molecules. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material.
Applications 6. Molecular weight determination Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds. For example, if we want to determine the molecular weight of amine then it is converted in to amine picrate.
Flame Emission Spectrophotometer PRINCIPLE: Atoms of many metallic element, when given sufficient energy, emit this energy at a wave length characteristic for that element. A specific amount of energy is absorbed by an orbital electron. These electrons being unstable in high energy state, release this energy as photons of a particular wavelength.
Flame Emission Spectrophotometer Used for the quantitative measurement of sodium and potassium in the body fluids. Also used for measurement of lithium, usually used in the treatment of psychiatric disorders, for therapeutic monitoring.
Summary 45 The spectrophotometer technique is to measures light intensity as a function of wavelength. It does this by: 1. diffracting the light beam into a spectrum of wavelengths 2. direct it to an object 3. receiving the light reflected or returned from the object 4. detecting the intensities with a charge-coupled device 5. displaying the results as a graph on the detector and then the display device.[1],[2]
Summary Applications Quantitative estimation of biological constituents Detection of functional groups Detection of impurities Molecular weight determination
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